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Cathepsins and cystatin C in atherosclerosis and obesity
Biochimie 92 (2010) 1580e1586
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Review
Cathepsins and cystatin C in atherosclerosis and obesity Jean-Charles Lafarge a, b, c, Nadia Naour a, b, c, Karine Clément a, b, c, d, e, Michèle Guerre-Millo a, b, c, * a
INSERM, U872, Eq7 Nutriomique, Paris, F-75006 France Université Pierre et Marie Curie-Paris6, Centre de Recherche des Cordeliers, UMR S 872, Paris, F-75006 France c Université Paris Descartes-Paris5, UMR S 872, Paris, F-75006 France d Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Nutrition and Endocrinology, Paris, F-75013 France e Center of Research on Human Nutrition Ile de France, Paris, F-75013 France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2010 Accepted 16 April 2010 Available online 24 April 2010
Given the increasing prevalence of human obesity worldwide, there is an urgent need for a better understanding of the molecular mechanisms linking obesity to metabolic and cardiovascular diseases. Our knowledge is nevertheless limited regarding molecules linking adipose tissue to downstream complications. The importance of cathepsins was brought to light in this context. Through a large scale transcriptomic analysis, our group recently identified the gene encoding cathepsin S as one of the most deregulated gene in the adipose tissue of obese subjects and positively correlated with body mass index. Other members of the cathepsin family are expressed in the adipose tissue, including cathepsin K and cathepsin L. Given their implication in atherogenesis, these proteases could participate into the well established deleterious relationship between enlarged adipose tissue and increased cardiovascular risk. Here, we review the clinical and experimental evidence relevant to the role of cathepsins K, L and S and their most abundant endogenous inhibitor, cystatin C, in atherosclerosis and in obesity. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Cathepsins Cystatin C Atherosclerosis Obesity
1. Introduction In the past decade, our vision of adipose tissue has changed considerably. A new concept has emerged that positions the adipose tissue as a major player in the ethiopathogenicity of metabolic and vascular co-morbidities of obesity. The enlarged adipose tissue of obese subjects is not only characterized by an excess of fat storage but also by a deregulation of adipose secretory functions. Adipose tissue produces a vast panel of biomolecules, including pro-inflammatory factors (IL-6, IL-1, MCP-1) and prothrombotic mediators (PAI-1), whose circulating levels are increased in obesity [1e3]. These factors originate from enlarged adipose cells and/or from the stromal inflammatory cells, mainly macrophages that massively accumulate in the adipose tissue in obese subjects [4e7]. Our team has previously reported that the severity of hepatic fibro-inflammation increased with macrophages accumulation in the visceral fat depot of morbidly obese patients [5,8], linking abundance of adipose tissue macrophages to a comorbidity of obesity. The pathophysiological relevance of adiposederived pro-inflammatory factors is incompletely understood and new adipose-secreted biomolecules are likely to be discovered.
* Corresponding author. INSERM U872 Eq 7, Centre de Recherche des Cordeliers, 15 Rue de l’Ecole de Médecine, 75006 Paris, France. Tel.: þ33 1 42 34 69 56. E-mail address: [email protected] (M. Guerre-Millo). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.04.011
The emerging role of new effective candidates belonging to the cathepsin family of cysteine proteases was recently brought to light. The functional proprieties of these proteases arouse much interest in recent years due to their therapeutic potential. It is well known that some of these proteases, including cathepsin S and cathepsin L, are important participants in antigen processing and presentation. The roles of cathepsins are not limited to the antigen presentation, as demonstrated in various diseases models [9]. In the present review, we will focus on three cathepsins, cathepsins K, L and S, and highlight the significance of their potential implication in atherosclerosis and obesity. Additionally, since cathepsins activity is regulated by endogenous inhibitors, the most abundant being cystatin C, we will also address the role of this protein in these pathologies. 2. Cathepsins K, L and S in atherosclerosis Extra cellular matrix proteins such as elastin and collagens contribute to the structural integrity of the vascular wall. The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. Different families of proteolytic enzymes may participate in this process, including matrix metalloproteinases, serine proteases and cathepsin cysteine proteases [10e12]. In this section, we discuss the role of selected cathepsin family members in the pathogenesis of
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atherosclerotic plaque formation and stability in humans and in genetically created mice models of atherogenesis. 2.1. Cathepsins K, L and S in human atherosclerotic lesions Cathepsin K and cathepsin S were the first cathepsins found to be expressed in human atheroma more than a decade ago [13]. While normal arteries contained little or no cathepsin K or cathepsin S, the proteins are abundantly expressed in macrophages and smooth muscle cells (SMC) of the atherosclerotic lesions. Early lesions show cathepsin K expression in the intimal and medial SMC, while, in advanced atherosclerotic plaques, cathepsin K localized mainly in macrophages and SMC of the fibrous cap. In addition, immunohistochemical analysis of atherosclerotic coronary arteries showed a positive correlation between cathepsin K in endothelium and internal elastic lamina breaks [14]. Beside cathepsin S and cathepsin K, increased expression of cathepsin L was reported in SMC, endothelial cells and macrophages of human aortic abdominal aneurysm and advanced atherosclerotic lesions [15]. Cell culture experiments showed that pro-inflammatory mediators, including IL-1beta, IFN-gamma and TGF beta, stimulate cathepsin S and cathepsin L gene expression and increased elastolytic and collagenolytic activity in human aortic SMC and macrophages [13,15,16]. This suggests that inflammatory processes that prevail during plaque formation increase locally the presence of active form of these cathepsins. The ability of SMC and macrophages to use cathepsins to degrade elastin and collagen support a role for these proteases in vessel wall alterations in humans.
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Table 1 Cathepsin K, L and S and cystatin C in atherosclerosis: vascular phenotype of knockout mice on a pro-atherogenic genetic background. Genotype
Vascular phenotype
References
Cat K//ApoE/
Y Y [ Y [ [
[17,18,60]
CatK/ leukocytes in LDLR/
¼ Plaque size YY Collagen content Y Elastin breaks [ Macrophages
[19]
Cat L//LDLR/
Y Y Y Y
Plaque size Collagen content Elastin breaks Macrophages, CD4þ cells and SMC
[21]
Cat S//LDLR/
Y Y Y Y Y
Plaque size Plaque progression Collagen content Elastin breaks Macrophages, CD4þ cells and SMC
[22]
Cat S//ApoE/
Y Plaque size [[ Plaque stability
[23]
CysC//ApoE/
[ [ [ [ [
[49,50]
Plaque size Plaque progression Collagen content Elastin breaks Macrophage foam cell formation Plaque stability
or ¼ Plaque size Collagen content Elastin breaks Macrophage content SMC
SMC: smooth muscle cells.
2.2. Cathepsins K, L and S in knockout mice models Direct evidence for cathepsins implication in atherogenesis was investigated in established mice models of atherogenesis, i.e., mice deleted for the low-density lipoprotein receptor (LDLR/) or mice deleted for the apolipoprotein E (ApoE/). Cross-breeding mice deficient in cathepsin K (CatK/), cathepsin L (CatL/) or cathepsin S (CatS/) with these atherosclerosis-prone mice allowed investigating the influence of cathepsin deficiency on plaque development and composition. The vascular phenotypes of the various models published so far are summarized in Table 1. Cathepsin K deficiency in ApoE/ mice substantially reduced plaque area owing to a decrease in the number of advanced lesions as well as a decrease in the size of plaque surface [17]. Advanced plaques of the double knockout mice showed an increase in collagen content and were less prone to rupture than those of ApoE/ mice. However, in the absence of cathepsin K, macrophage size was increased because of the abundant cellular storage of cholesterol esters. In another study, the effect of cathepsin K deficiency was investigated in the brachiocephalic arteries of ApoE/ mice fed a cholate-containing high fat diet [18]. On this diet, ApoE/ mice displayed severe lesions with buried fibrous caps, and are considered a model of human plaque vulnerability with risk of rupture. In absence of cathepsin K, the CatK//ApoE/ mice showed a significantly decreased number of buried fibrous caps, increased collagen content, smaller plaque areas and maintained elastic lamina integrity. Thus, deficiency in cathepsin K reduced plaque progression and induced plaque fibrosis, potentially favoring stability, but aggravated macrophage foam cell formation in these models. More recently, the role of cathepsin K in vessel wall remodeling was re-assessed in a distinct mice model in which cathepsin K was selectively disrupt in the hematopoietic system by the technique of bone marrow transplantation in LDLR/ mice [19]. As in cathepsin K total knockout mice, the absence of cathepsin K in leukocytes resulted in less elastic lamina fragmentation in the atheroslerotic lesions of LDLR/ mice, although, in this model,
lesion size was not affected. However, in the absence of leukocyte cathepsin K, collagen content was dramatically reduced, instead of being increased. This was attributed to diminished migration of collagen-producing SMC into the plaque because of enhanced elastic lamina integrity. Moreover, the number of macrophages was increased in atherosclerotic lesions, potentially contributing to plaque destabilization. Thus, at present, it is not clear whether cathepsin K deficiency is beneficial or detrimental to atherosclerotic plaque stability [20]. The lack of cathepsin L in CatL//LDLR/ double knockout mice resulted in significantly smaller atherosclerotic lesions compared with LDLR/ mice [21]. In addition, the lesions showed significantly reduced levels of collagen and less medial elastin degradation. The number of CD4þ T-lymphocytes, macrophages and SMC was also reduced. Interestingly, the SMC obtained from CatL//LDLR / mice demonstrated low capacity to degrade elastin or collagen and to transmigrate through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix. These findings indicate that cathepsin L promotes several processes involved in plaque progression, particularly SMC and macrophages migration into the atherosclerotic plaque. The effects of cathepsin S deficiency were investigated on both the LDLR/ and ApoE/ genetic backgrounds. In the first published model, CatS//LDLR/ double knockout mice showed a reduction in the size of atherosclerotic plaque and stage of development [22]. Aortas from these mice had preserved elastic lamina with a reduction in the number of elastin breaks. These mice also displayed markedly reduced number of intimal macrophages, CD4þ T lymphocytes and SMC, and decreased collagen content and fibrous cap thickness. In the second study, CatS//ApoE/ mice displayed smaller atherosclerotic plaques in the brachiocephalic arteries with markedly less plaque ruptures [23]. The number of
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buried fibrous layers, indicative of an unstable plaque phenotype, was substantially reduced in the double knockouts. Altogether, these data provide evidence for a role of cathepsin S in atherosclerosis, favoring the progression and plaque destabilization. To conclude, human and animal data strongly support the idea that cathepsins K, L and S contribute to the formation and progression of atherosclerotic plaques and may also participate into the destabilization of advanced plaques. The cellular and molecular mechanisms, which might vary for each cathepsin, and the functional hierarchy among cathepsin family members and other extracellular matrix proteases, remain to be established. Finally, whether inhibition of selected cathepsins might be beneficial in a treatment strategy for atherosclerosis and plaque stabilization has yet to be investigated. 3. Cathepsins K, L and S in obesity Identifying the molecules linking inflamed adipose tissue to downstream complications is a challenging area of ongoing research. A strategy is to look for genes whose expression is related quantitatively to body mass index (BMI) or fat mass. Through a large scale transcriptomic analysis, we have found 240 genes significantly overexpressed in the adipose tissue of morbidly obese subjects compared to non-obese controls. Cathepsin S gene was the first named gene in the list which was highly correlated with BMI and decreased after gastric surgery-induced weight loss [24,25]. Moreover, data from our and other groups suggest that cathepsin K and cathepsin L are also expressed in human adipose tissue depots [24,26e28]. In this section, we will review the potential role of these three cathepsins in obesity. 3.1. Cathepsins K, L and S in human obesity Given the presence of several cathepsin family members in adipose tissue, it was of interest to identify the most clinically relevant cathepsin(s) in human obesity. To address this point, we designed a clinical study in which adipose expression and circulating concentrations of cathepsins S, L and K were investigated in the same subject [29]. This allowed comparing the effect of changes in energy balance on each cathepsin, without confounding factors such as nutritional, metabolic and/or hormonal variations. We included a group of massively obese women undergoing gastric surgery and a group of moderately obese women submitted to medically-supervised caloric restriction. First, we showed that cathepsins K, L and S are indeed highly expressed in subcutaneous and visceral adipose tissue compared to other tissues (liver, muscle and jejunum) of the same subject. Regarding cathepsin K, we found that the protein, known to be mostly localized in lysosomes [10,30], was undetectable in the circulation and in the medium of adipose tissue explants, indicating that this cathepsin is virtually not secreted. Cathepsin K gene expression in adipose tissue was not significantly altered by obesity and weight reduction. Although cathepsin L was measurable in serum, we found no significant difference in circulating concentrations between lean and obese subjects before or after weight loss. In line with these data, similar levels of cathepsin L expression and protein release by adipose tissue explants were observed in lean and obese subjects. By contrast, obesity significantly increased cathepsin S in adipose tissue and in circulation, confirming our previous observations [24,25]. Unlike cathepsin L, cathepsin S was released in increased amounts in adipose tissue of obese subjects. Weight reduction reduced circulating cathepsin S concentrations in the two clinical settings investigated in this study. This occurred despite markedly distinct amounts of weight loss (i.e., e 15% after gastric surgery versus e 5% after caloric restriction). This suggests that reduction of
serum cathepsin S might be more related to decreased energy intake than to diminution of body fat mass. According to these results, it appears that cathepsin S is more influenced than cathepsins K and L by changes in energy balance known to markedly influence metabolic risks in humans. These observations suggest that cathepsin S is the clinically relevant cathepsin in human obesity, although potential implication of other cathepsins, not yet investigated in this context, cannot be ruled out. 3.2. Cathepsins K and L knockout mice models The effects of cathepsin K and cathepsin L deficiency on energy homeostasis have been investigated in genetically modified mice (Table 2). In these studies, mice were fed either a low fat or a high fat diet that recapitulates some of the alterations seen in common human obesity, including adipose tissue enlargement, dyslipidemia, hyperinsulinemia and glucose intolerance. Two groups have investigated the phenotype of CatK/ mice, with slight discrepancies. In a first model, Funicello et al. [31] found that young male and female CatK/ mice displayed significantly lower body weight and adipose tissue mass as compared to wild-type mice between 5 and 8 weeks of age, while no difference was observable in adult mice (12e20 weeks). Circulating levels of insulin and glucose response to an intraperitoneal glucose tolerance test were lower in adult CatK/ mice, where no body weight difference was visible. When submitted to a high fat nutritional challenge, CatK/ mice gained significantly less weight than controls and showed a lower percentage of body fat. Plasma triglycerides and cholesterol were significantly lower in male CatK/ mice (not in females), but no major amelioration of hyperinsulinemia or blood glucose were observed compared to high fat fed wild-type mice. In a second study, Yang et al. [28] report the phenotype of CatK/ mice fed a high fat diet, showing that cathepsin K deficiency reduced body weight gain, with a larger effect in female than in male mice. Reduced body weight gain was accompanied by a reduction in fat pads weight in mice of both genders, in line with the previous study. However, lower levels of insulin were detected in the serum of CatK/ mice along with unchanged (male) or reduced serum glucose (female). These data identify cathepsin K as a new player in the control of body weight gain and fat accretion in
Table 2 Cathepsin K and L in obesity: metabolic phenotype of knockout mice or mice treated with specific inhibitors. Genotype
Chow diet
Cat K/
Y Body weight (young) or Y Body weight [28,31] (female) ¼ (adult) Y Adipose tissue mass Y Serum insulin Y or ¼ Serum insulin ¼ Serum glucose Y or ¼ Serum glucose
C57BL/6 þK4b ob/ob þK4b
Y Body weight (female)
Cat L/
Y Body weight after 7 weeks of age Y Serum insulin Y Serum glucose [ Glucose tolerance
ob/ob þCLIK195
Y Body weight Y Serum insulin [ Glucose tolerance
High fat diet
References
Y Body weight (female)
[28]
Y Body weight
[27]
[28]
Y Y Y [
Adipose tissue mass Serum insulin Serum glucose Glucose tolerance [27]
K4b: Na-phenoxybenzyloxycarbonyl-L-leucine (2-phenylaminoethyl) amide: cathepsin K-selective inhibitor; CLIK195: cathepsin L-selective inhibitor. ob/ob mice are genetically obese due to the lack of leptin.
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condition of high fat feeding. This protease might also contribute to the development of dyslipidemia, glucose intolerance and insulin resistance. Of note, in both studies, cathepsin K deficiency did not promote major changes in food intake and energy expenditure, suggesting a peripheral rather than a central effect of cathepsin K. The metabolic phenotype of cathepsin L deficient mice has been investigated as well [27]. Young CatL/ mice have normal body weight until 7 weeks of age, but show markedly reduced body weight gain thereafter, both on low or high fat diet. In high fat fed CatL/mice, body weight reduction occurred in conjunction with reduced fat mass, although food intake and energy expenditure was no less than in wild-type littermates. Reduced levels of serum glucose and insulin and increased glucose tolerance in glucose tolerance testing were also apparent in this model. Interestingly, the improvement of glucose tolerance and serum insulin in CatL/ mice appears be related to an effect of cathepsin L deletion per se, since the phenotype was present in 6-week old CatL/ mice that had not developed significant differences in body weight and fat deposition from control mice. In conclusion, these studies revealed that mice deficient in cathepsin K or in cathepsin L display a lean phenotype and show features of improved glucose homeostasis, partly independent of their reduced fat mass. This beneficial metabolic phenotype persists when mice are challenged by a high fat diet. Thus, in addition to a pro-atherogenic effect, these cathepsins might also contribute to the alterations of glucose and lipid homeostasis seen in obesity. Additionally, since our data in humans point out cathepsin S as clinically relevant, the metabolic phenotype of mice deficient in cathepsin S, that has not been reported so far, could shed new light on the implication of this cathepsin in human obesity. 3.3. Cathepsins K, L and S and adipose differentiation The mechanisms linking cathepsins protease activity to adipose tissue growth are yet to be fully deciphered. Cellular experiments performed in 3T3-L1 murine adipocyte cell line show that cathepsin K gene expression increases over adipose differentiation and that the E64 cathepsin K inhibitor reduces triglyceride accumulation [26]. Other studies investigated the regulation of cathepsins in primary human pre-adipocytes, showing that all three cathepsins K, L and S demonstrate a marked induction after differentiation [27,28,32]. When the human pre-adipocytes were exposed to selective cathepsins inhibitors, they fail to differentiate properly, as evidenced by reduced lipid accumulation. This suggests a pro-adipogenic effect of these proteases, which has been attributed to their capacity to degrade extracellular matrix proteins, mainly fibronectin whose degradation is required for adipogenesis [33]. Altogether, these observations are in line with the lean phenotype of CatK/ and CatL/ mice and suggest that increased cathepsins levels facilitate adipogenesis and/or adipose cell hypertrophy in the adipose tissue of obese subjects, at least in part through extracellular matrix remodelling. 3.4. Mice treated with cathepsin K and cathepsin L pharmacological inhibitors The anti-adipogenic effects of cathepsins deletion in mice and in adipose cell culture experiments prompted to investigate the effect of cathepsins inhibitors in vivo. The phenotypes induced in mice by treatments with cathepsin K [28] or cathepsin L inhibitors [27] are summarized in Table 2. The cathepsin K inhibitor Na-phenoxybenzyloxycarbonyl-L-leucine (2-phenylaminoethyl) amide (K4b) inhibits recombinant human cathepsin K with IC50 < 0.006 mM, whereas it inhibits recombinant human cathepsins S and L with
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IC50 >9.5 and >10 mM, respectively [34]. When administered to high fat fed female mice (ip, 1 mg/kg/day), this inhibitor induced a significant reduction of body weight gain. A similar effect of weight reduction was obtained in genetically obese ob/ob mice that received K4b at 4 weeks of age for 8 weeks. In agreement with the lack of effect of cathepsin K deletion, all tested energy expenditure parameters, including food intake, O2 consumption, CO2 production, and metabolic rate were not affected in wild-type or ob/ob mice by this compound [28]. Of note, K4b might not be very selective in mice. Indeed, a recent study indicates that this compound has a greater potency to inhibit mouse cathepsins S and L than mouse cathepsin K [35]. Therefore, some of the weightreducing effects of 4Kb reported in mice models of obesity might result in part from reduction of cathepsin S and/or cathepsin L activity. The pharmacological inhibition of cathepsin L was performed using CLIK195 on both diet-induced and genetically obese ob/ob mice [27]. Mice were injected intraperitoneally with 100 mg/kg/day of the compound. Compared to vehicle controls, this inhibitor reduced moderately body weight gain in male mice fed a high fat diet and had no significant effect on food intake and glycemic parameters. The same dose of CLIK195, however, demonstrated profound and gender-independent effect in reducing body weight gain, serum insulin levels and glucose intolerance in ob/ob mice, although food consumption and energy expenditure were not substantially altered. If transposable in humans, these data suggest a novel therapeutic strategy for the control of obesity by regulating cathepsin K and/or cathepsin L activities. Because of the known negative effect of cathepsin K on bone formation, several inhibitors have been tested for treating osteoporosis, while cathepsin S inhibitors are in development to treat autoimmune diseases, such as psoriasis, asthma and rheumatoid arthritis [36,37]. Currently, there is no data for their use in countering obesity-linked atherosclerosis and/or metabolic dysfunction in humans. A prerequisite for therapeutic use of pharmacological cathepsins inhibitors is a better understanding of the mechanisms relying cathepsins enzymatic activity to metabolic and inflammatory pathways, specifically in the adipose tissue and at the site of vascular wall alteration. Moreover, it is established that the enzymatic activity of cysteine proteases is regulated by endogenous inhibitors belonging to the cystatin superfamily (see below). How pharmacological inhibition could interfere with endogenous inhibition of cathepsins either locally or systemically, has to be determined. 4. Cystatin C in atherosclerosis The cystatin superfamily comprises a large group of proteins present in a wide variety of organisms, including humans [38e40]. Cystatin inhibitory activity is vital for the regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases, including cysteine cathepsins. Cystatin C, a small protein produced by virtually all organs and found in biological fluids including blood, is the most abundant and potent endogenous inhibitor of cathepsins. Its implication in clinical and experimental atherogenesis has therefore been investigated by several groups. 4.1. Cystatin C in atherogenesis In humans, cystatin C protein is detected in normal arteries, but severely reduced in atherosclerotic and aneurismal aortic lesions where cathepsins K and S are abundant [41]. Moreover, reduced serum cystatin C concentrations were reported in patients with abdominal aortic aneurysms [41,42]. These observations support an
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anti-atherogenic role of cystatin C. Contrary to this hypothesis, epidemiological studies consistently report a positive relationship between elevated circulating cystatin C and cardiovascular outcomes in selected populations, including elderly participants and patients with established vascular diseases or increased cardiovascular risk [43e48]. This counterintuitive association suggests that elevation of cystatin C represents a compensatory mechanism to reduce pro-atherogenic cathepsins activity. 4.2. Cystatin C knockout mice models To assess directly the effect of cystatin C on plaque formation, two groups have investigated the vascular phenotype of double ApoE- cystatin C knockout mice (ApoE //CysC /), with slightly different results (Table 1). Sukhova et al. [49] found that cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation and increased SMC accumulation, but no difference in the size of aortic lesions induced by an atherogenic high-cholesterol diet. Of note, collagen content was increased and not reduced, as would be expected after deletion of the cathepsin inhibitor. In the other study, Bengtsson et al. [50] report increased plaque size and macrophages content in the double knockout mice when fed a high fat diet. These findings demonstrate directly the importance of cystatin C in deregulated arterial integrity during experimental atherogenesis. However, the cellular and molecular mechanisms involved, either direct or through cathepsins inhibition, remain to be established. 5. Cystatin C in human obesity Unrelated to its functional role as an inhibitor of cathepsins, cystatin C has emerged as a surrogate marker of the glomerular filtration rate (GFR) at least as good as serum creatinine [51,52]. Indeed, the protein is freely filtered by the renal glomeruli and metabolized by the proximal tubule. However, unlike creatinine, which reflects muscle mass, the main tissue source of cystatin C was unknown. A few studies have reported that increased adiposity or obesity associate with higher cystatin C circulating concentrations. Positive associations of serum cystatin C with BMI were found in a geriatric population [53], in the general population [45], and, with body weight, in a large Caucasian population [54]. Higher waist circumference and increased per cent of body fat also associated with higher serum cystatin C in apparently healthy subjects [55,56]. Recently, a graded association between higher BMI and elevated serum cystatin C was reported in American adults [57]. In these epidemiological studies, however, the contribution of increased adiposity versus reduced GFR in obesity-linked elevation of serum cystatin C was unclear. To address this point, we measured serum cystatin C in a large population of non-obese and obese subjects [58]. Using the abbreviated equation from the Modification of Diet in Renal Disease (MDRD) study [59], we estimated systematically GFR in all participants. This allowed measuring the respective effect of obesity and reduced GFR to increase the circulating concentrations of cystatin C. Interestingly we found that circulating cystatin C was consistently elevated in obese subjects of both genders, independently of reduced eGFR. These observations strongly suggest a role for adipose tissue as a contributor to circulating concentrations of this protein in obesity. In support of this hypothesis, we showed that adipose tissue expression of cystatin C is increased in obesity and the protein abundantly secreted by human adipose tissue explants in vitro, mainly by stroma vascular cells that express high levels of cystatin C. These data suggest that adipose tissue production of cystatin C contributes to the elevation
of circulating concentrations in obesity, although the participation of other cells or tissues where cystatin C is expressed cannot be excluded. The consequences of elevated serum cystatin C in human obesity remain largely hypothetical. Increased serum cystatin C might be part of regulatory mechanisms engaged to control the pro-atherogenic capacity of specific cathepsins. As indicated by the lean phenotype of CatK/ and CatL/ mice, certain cathepsins might promote fat accretion. In this context, increased adipose cystatin C could exert a protective effect to control adipose tissue enlargement through cathepsins inhibition. Although further studies are required to decipher the actual pathophysiological roles of cystatin C, this protein can be added to the list of adiposesecreted factors with the potential to affect adipose tissue biology and obesity-linked complications through local and/or systemic actions. 6. Conclusion In obesity, adipose tissue secretes numerous bioactive molecules that may be acting synergistically in developing cardiovascular diseases and other complications associated with the deregulation of glucose and lipid homeostasis. As reviewed here, members of the cathepsin family, including cathepsins K, L and S, are among the newly discovered potential culprits. This opens new avenues to test whether individual or combined inhibition of selected cathepsins could reduce cardiovascular risk and ameliorate metabolic status in obese subjects. Given the current development of cathepsins inhibitors for diverse pathologies, these drugs could be tested in the near future in obese patients. Nevertheless, further clinical and experimental studies are obviously needed to establish the proof of concept that cathepsins inhibition is beneficial in obesity metabolic and cardiovascular comorbidities. Acknowledgments The authors thanks supports from Clinical Research Contracts (Assistance Publique/Direction de la Recherche Clinique, AOR 02076) for human studies. This work was supported by a grant from the European Community (Collaborative Project ADAPT, HEALTHF262008-201100), a grant from the French National Agency of Research (OBCAT, program ANR-05-PCOD-026-01), a prize DANONE/Fondation pour la Recherche Médicale. References [1] M. Guerre-Millo, Adipose tissue and adipokines: for better or worse. Diabetes Metab. 30 (2004) 13e19. [2] P. Trayhurn, I.S. Wood, Signalling role of adipose tissue: adipokines and inflammation in obesity. Biochem. Soc. Trans. 33 (2005) 1078e1081. [3] P. Trayhurn, Adipocyte biology. Obes. Rev. 8 (Suppl. 1) (2007) 41e44. [4] R. Cancello, C. Henegar, N. Viguerie, S. Taleb, C. Poitou, C. Rouault, M. Coupaye, V. Pelloux, D. Hugol, J.L. Bouillot, A. Bouloumie, G. Barbatelli, S. Cinti, P.A. Svensson, G.S. Barsh, J.D. Zucker, A. Basdevant, D. Langin, K. Clement, Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgeryinduced weight loss. Diabetes 54 (2005) 2277e2286. [5] R. Cancello, J. Tordjman, C. Poitou, G. Guilhem, J.L. Bouillot, D. Hugol, C. Coussieu, A. Basdevant, H.A. Bar, P. Bedossa, M. Guerre-Millo, K. Clement, Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity. Diabetes 55 (2006) 1554e1561. [6] C.A. Curat, V. Wegner, C. Sengenes, A. Miranville, C. Tonus, R. Busse, A. Bouloumie, Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. Diabetologia 49 (2006) 744e747. [7] I. Harman-Boehm, M. Bluher, H. Redel, N. Sion-Vardy, S. Ovadia, E. Avinoach, I. Shai, N. Kloting, M. Stumvoll, N. Bashan, A. Rudich, Macrophage infiltration into omental versus subcutaneous fat across different populations: effect of
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[17]
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[27]
[28]
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Review
Cathepsins and cystatin C in atherosclerosis and obesity Jean-Charles Lafarge a, b, c, Nadia Naour a, b, c, Karine Clément a, b, c, d, e, Michèle Guerre-Millo a, b, c, * a
INSERM, U872, Eq7 Nutriomique, Paris, F-75006 France Université Pierre et Marie Curie-Paris6, Centre de Recherche des Cordeliers, UMR S 872, Paris, F-75006 France c Université Paris Descartes-Paris5, UMR S 872, Paris, F-75006 France d Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Nutrition and Endocrinology, Paris, F-75013 France e Center of Research on Human Nutrition Ile de France, Paris, F-75013 France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2010 Accepted 16 April 2010 Available online 24 April 2010
Given the increasing prevalence of human obesity worldwide, there is an urgent need for a better understanding of the molecular mechanisms linking obesity to metabolic and cardiovascular diseases. Our knowledge is nevertheless limited regarding molecules linking adipose tissue to downstream complications. The importance of cathepsins was brought to light in this context. Through a large scale transcriptomic analysis, our group recently identified the gene encoding cathepsin S as one of the most deregulated gene in the adipose tissue of obese subjects and positively correlated with body mass index. Other members of the cathepsin family are expressed in the adipose tissue, including cathepsin K and cathepsin L. Given their implication in atherogenesis, these proteases could participate into the well established deleterious relationship between enlarged adipose tissue and increased cardiovascular risk. Here, we review the clinical and experimental evidence relevant to the role of cathepsins K, L and S and their most abundant endogenous inhibitor, cystatin C, in atherosclerosis and in obesity. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Cathepsins Cystatin C Atherosclerosis Obesity
1. Introduction In the past decade, our vision of adipose tissue has changed considerably. A new concept has emerged that positions the adipose tissue as a major player in the ethiopathogenicity of metabolic and vascular co-morbidities of obesity. The enlarged adipose tissue of obese subjects is not only characterized by an excess of fat storage but also by a deregulation of adipose secretory functions. Adipose tissue produces a vast panel of biomolecules, including pro-inflammatory factors (IL-6, IL-1, MCP-1) and prothrombotic mediators (PAI-1), whose circulating levels are increased in obesity [1e3]. These factors originate from enlarged adipose cells and/or from the stromal inflammatory cells, mainly macrophages that massively accumulate in the adipose tissue in obese subjects [4e7]. Our team has previously reported that the severity of hepatic fibro-inflammation increased with macrophages accumulation in the visceral fat depot of morbidly obese patients [5,8], linking abundance of adipose tissue macrophages to a comorbidity of obesity. The pathophysiological relevance of adiposederived pro-inflammatory factors is incompletely understood and new adipose-secreted biomolecules are likely to be discovered.
* Corresponding author. INSERM U872 Eq 7, Centre de Recherche des Cordeliers, 15 Rue de l’Ecole de Médecine, 75006 Paris, France. Tel.: þ33 1 42 34 69 56. E-mail address: [email protected] (M. Guerre-Millo). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.04.011
The emerging role of new effective candidates belonging to the cathepsin family of cysteine proteases was recently brought to light. The functional proprieties of these proteases arouse much interest in recent years due to their therapeutic potential. It is well known that some of these proteases, including cathepsin S and cathepsin L, are important participants in antigen processing and presentation. The roles of cathepsins are not limited to the antigen presentation, as demonstrated in various diseases models [9]. In the present review, we will focus on three cathepsins, cathepsins K, L and S, and highlight the significance of their potential implication in atherosclerosis and obesity. Additionally, since cathepsins activity is regulated by endogenous inhibitors, the most abundant being cystatin C, we will also address the role of this protein in these pathologies. 2. Cathepsins K, L and S in atherosclerosis Extra cellular matrix proteins such as elastin and collagens contribute to the structural integrity of the vascular wall. The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves substantial proteolysis of the arterial extracellular matrix. Different families of proteolytic enzymes may participate in this process, including matrix metalloproteinases, serine proteases and cathepsin cysteine proteases [10e12]. In this section, we discuss the role of selected cathepsin family members in the pathogenesis of
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atherosclerotic plaque formation and stability in humans and in genetically created mice models of atherogenesis. 2.1. Cathepsins K, L and S in human atherosclerotic lesions Cathepsin K and cathepsin S were the first cathepsins found to be expressed in human atheroma more than a decade ago [13]. While normal arteries contained little or no cathepsin K or cathepsin S, the proteins are abundantly expressed in macrophages and smooth muscle cells (SMC) of the atherosclerotic lesions. Early lesions show cathepsin K expression in the intimal and medial SMC, while, in advanced atherosclerotic plaques, cathepsin K localized mainly in macrophages and SMC of the fibrous cap. In addition, immunohistochemical analysis of atherosclerotic coronary arteries showed a positive correlation between cathepsin K in endothelium and internal elastic lamina breaks [14]. Beside cathepsin S and cathepsin K, increased expression of cathepsin L was reported in SMC, endothelial cells and macrophages of human aortic abdominal aneurysm and advanced atherosclerotic lesions [15]. Cell culture experiments showed that pro-inflammatory mediators, including IL-1beta, IFN-gamma and TGF beta, stimulate cathepsin S and cathepsin L gene expression and increased elastolytic and collagenolytic activity in human aortic SMC and macrophages [13,15,16]. This suggests that inflammatory processes that prevail during plaque formation increase locally the presence of active form of these cathepsins. The ability of SMC and macrophages to use cathepsins to degrade elastin and collagen support a role for these proteases in vessel wall alterations in humans.
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Table 1 Cathepsin K, L and S and cystatin C in atherosclerosis: vascular phenotype of knockout mice on a pro-atherogenic genetic background. Genotype
Vascular phenotype
References
Cat K//ApoE/
Y Y [ Y [ [
[17,18,60]
CatK/ leukocytes in LDLR/
¼ Plaque size YY Collagen content Y Elastin breaks [ Macrophages
[19]
Cat L//LDLR/
Y Y Y Y
Plaque size Collagen content Elastin breaks Macrophages, CD4þ cells and SMC
[21]
Cat S//LDLR/
Y Y Y Y Y
Plaque size Plaque progression Collagen content Elastin breaks Macrophages, CD4þ cells and SMC
[22]
Cat S//ApoE/
Y Plaque size [[ Plaque stability
[23]
CysC//ApoE/
[ [ [ [ [
[49,50]
Plaque size Plaque progression Collagen content Elastin breaks Macrophage foam cell formation Plaque stability
or ¼ Plaque size Collagen content Elastin breaks Macrophage content SMC
SMC: smooth muscle cells.
2.2. Cathepsins K, L and S in knockout mice models Direct evidence for cathepsins implication in atherogenesis was investigated in established mice models of atherogenesis, i.e., mice deleted for the low-density lipoprotein receptor (LDLR/) or mice deleted for the apolipoprotein E (ApoE/). Cross-breeding mice deficient in cathepsin K (CatK/), cathepsin L (CatL/) or cathepsin S (CatS/) with these atherosclerosis-prone mice allowed investigating the influence of cathepsin deficiency on plaque development and composition. The vascular phenotypes of the various models published so far are summarized in Table 1. Cathepsin K deficiency in ApoE/ mice substantially reduced plaque area owing to a decrease in the number of advanced lesions as well as a decrease in the size of plaque surface [17]. Advanced plaques of the double knockout mice showed an increase in collagen content and were less prone to rupture than those of ApoE/ mice. However, in the absence of cathepsin K, macrophage size was increased because of the abundant cellular storage of cholesterol esters. In another study, the effect of cathepsin K deficiency was investigated in the brachiocephalic arteries of ApoE/ mice fed a cholate-containing high fat diet [18]. On this diet, ApoE/ mice displayed severe lesions with buried fibrous caps, and are considered a model of human plaque vulnerability with risk of rupture. In absence of cathepsin K, the CatK//ApoE/ mice showed a significantly decreased number of buried fibrous caps, increased collagen content, smaller plaque areas and maintained elastic lamina integrity. Thus, deficiency in cathepsin K reduced plaque progression and induced plaque fibrosis, potentially favoring stability, but aggravated macrophage foam cell formation in these models. More recently, the role of cathepsin K in vessel wall remodeling was re-assessed in a distinct mice model in which cathepsin K was selectively disrupt in the hematopoietic system by the technique of bone marrow transplantation in LDLR/ mice [19]. As in cathepsin K total knockout mice, the absence of cathepsin K in leukocytes resulted in less elastic lamina fragmentation in the atheroslerotic lesions of LDLR/ mice, although, in this model,
lesion size was not affected. However, in the absence of leukocyte cathepsin K, collagen content was dramatically reduced, instead of being increased. This was attributed to diminished migration of collagen-producing SMC into the plaque because of enhanced elastic lamina integrity. Moreover, the number of macrophages was increased in atherosclerotic lesions, potentially contributing to plaque destabilization. Thus, at present, it is not clear whether cathepsin K deficiency is beneficial or detrimental to atherosclerotic plaque stability [20]. The lack of cathepsin L in CatL//LDLR/ double knockout mice resulted in significantly smaller atherosclerotic lesions compared with LDLR/ mice [21]. In addition, the lesions showed significantly reduced levels of collagen and less medial elastin degradation. The number of CD4þ T-lymphocytes, macrophages and SMC was also reduced. Interestingly, the SMC obtained from CatL//LDLR / mice demonstrated low capacity to degrade elastin or collagen and to transmigrate through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix. These findings indicate that cathepsin L promotes several processes involved in plaque progression, particularly SMC and macrophages migration into the atherosclerotic plaque. The effects of cathepsin S deficiency were investigated on both the LDLR/ and ApoE/ genetic backgrounds. In the first published model, CatS//LDLR/ double knockout mice showed a reduction in the size of atherosclerotic plaque and stage of development [22]. Aortas from these mice had preserved elastic lamina with a reduction in the number of elastin breaks. These mice also displayed markedly reduced number of intimal macrophages, CD4þ T lymphocytes and SMC, and decreased collagen content and fibrous cap thickness. In the second study, CatS//ApoE/ mice displayed smaller atherosclerotic plaques in the brachiocephalic arteries with markedly less plaque ruptures [23]. The number of
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buried fibrous layers, indicative of an unstable plaque phenotype, was substantially reduced in the double knockouts. Altogether, these data provide evidence for a role of cathepsin S in atherosclerosis, favoring the progression and plaque destabilization. To conclude, human and animal data strongly support the idea that cathepsins K, L and S contribute to the formation and progression of atherosclerotic plaques and may also participate into the destabilization of advanced plaques. The cellular and molecular mechanisms, which might vary for each cathepsin, and the functional hierarchy among cathepsin family members and other extracellular matrix proteases, remain to be established. Finally, whether inhibition of selected cathepsins might be beneficial in a treatment strategy for atherosclerosis and plaque stabilization has yet to be investigated. 3. Cathepsins K, L and S in obesity Identifying the molecules linking inflamed adipose tissue to downstream complications is a challenging area of ongoing research. A strategy is to look for genes whose expression is related quantitatively to body mass index (BMI) or fat mass. Through a large scale transcriptomic analysis, we have found 240 genes significantly overexpressed in the adipose tissue of morbidly obese subjects compared to non-obese controls. Cathepsin S gene was the first named gene in the list which was highly correlated with BMI and decreased after gastric surgery-induced weight loss [24,25]. Moreover, data from our and other groups suggest that cathepsin K and cathepsin L are also expressed in human adipose tissue depots [24,26e28]. In this section, we will review the potential role of these three cathepsins in obesity. 3.1. Cathepsins K, L and S in human obesity Given the presence of several cathepsin family members in adipose tissue, it was of interest to identify the most clinically relevant cathepsin(s) in human obesity. To address this point, we designed a clinical study in which adipose expression and circulating concentrations of cathepsins S, L and K were investigated in the same subject [29]. This allowed comparing the effect of changes in energy balance on each cathepsin, without confounding factors such as nutritional, metabolic and/or hormonal variations. We included a group of massively obese women undergoing gastric surgery and a group of moderately obese women submitted to medically-supervised caloric restriction. First, we showed that cathepsins K, L and S are indeed highly expressed in subcutaneous and visceral adipose tissue compared to other tissues (liver, muscle and jejunum) of the same subject. Regarding cathepsin K, we found that the protein, known to be mostly localized in lysosomes [10,30], was undetectable in the circulation and in the medium of adipose tissue explants, indicating that this cathepsin is virtually not secreted. Cathepsin K gene expression in adipose tissue was not significantly altered by obesity and weight reduction. Although cathepsin L was measurable in serum, we found no significant difference in circulating concentrations between lean and obese subjects before or after weight loss. In line with these data, similar levels of cathepsin L expression and protein release by adipose tissue explants were observed in lean and obese subjects. By contrast, obesity significantly increased cathepsin S in adipose tissue and in circulation, confirming our previous observations [24,25]. Unlike cathepsin L, cathepsin S was released in increased amounts in adipose tissue of obese subjects. Weight reduction reduced circulating cathepsin S concentrations in the two clinical settings investigated in this study. This occurred despite markedly distinct amounts of weight loss (i.e., e 15% after gastric surgery versus e 5% after caloric restriction). This suggests that reduction of
serum cathepsin S might be more related to decreased energy intake than to diminution of body fat mass. According to these results, it appears that cathepsin S is more influenced than cathepsins K and L by changes in energy balance known to markedly influence metabolic risks in humans. These observations suggest that cathepsin S is the clinically relevant cathepsin in human obesity, although potential implication of other cathepsins, not yet investigated in this context, cannot be ruled out. 3.2. Cathepsins K and L knockout mice models The effects of cathepsin K and cathepsin L deficiency on energy homeostasis have been investigated in genetically modified mice (Table 2). In these studies, mice were fed either a low fat or a high fat diet that recapitulates some of the alterations seen in common human obesity, including adipose tissue enlargement, dyslipidemia, hyperinsulinemia and glucose intolerance. Two groups have investigated the phenotype of CatK/ mice, with slight discrepancies. In a first model, Funicello et al. [31] found that young male and female CatK/ mice displayed significantly lower body weight and adipose tissue mass as compared to wild-type mice between 5 and 8 weeks of age, while no difference was observable in adult mice (12e20 weeks). Circulating levels of insulin and glucose response to an intraperitoneal glucose tolerance test were lower in adult CatK/ mice, where no body weight difference was visible. When submitted to a high fat nutritional challenge, CatK/ mice gained significantly less weight than controls and showed a lower percentage of body fat. Plasma triglycerides and cholesterol were significantly lower in male CatK/ mice (not in females), but no major amelioration of hyperinsulinemia or blood glucose were observed compared to high fat fed wild-type mice. In a second study, Yang et al. [28] report the phenotype of CatK/ mice fed a high fat diet, showing that cathepsin K deficiency reduced body weight gain, with a larger effect in female than in male mice. Reduced body weight gain was accompanied by a reduction in fat pads weight in mice of both genders, in line with the previous study. However, lower levels of insulin were detected in the serum of CatK/ mice along with unchanged (male) or reduced serum glucose (female). These data identify cathepsin K as a new player in the control of body weight gain and fat accretion in
Table 2 Cathepsin K and L in obesity: metabolic phenotype of knockout mice or mice treated with specific inhibitors. Genotype
Chow diet
Cat K/
Y Body weight (young) or Y Body weight [28,31] (female) ¼ (adult) Y Adipose tissue mass Y Serum insulin Y or ¼ Serum insulin ¼ Serum glucose Y or ¼ Serum glucose
C57BL/6 þK4b ob/ob þK4b
Y Body weight (female)
Cat L/
Y Body weight after 7 weeks of age Y Serum insulin Y Serum glucose [ Glucose tolerance
ob/ob þCLIK195
Y Body weight Y Serum insulin [ Glucose tolerance
High fat diet
References
Y Body weight (female)
[28]
Y Body weight
[27]
[28]
Y Y Y [
Adipose tissue mass Serum insulin Serum glucose Glucose tolerance [27]
K4b: Na-phenoxybenzyloxycarbonyl-L-leucine (2-phenylaminoethyl) amide: cathepsin K-selective inhibitor; CLIK195: cathepsin L-selective inhibitor. ob/ob mice are genetically obese due to the lack of leptin.
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condition of high fat feeding. This protease might also contribute to the development of dyslipidemia, glucose intolerance and insulin resistance. Of note, in both studies, cathepsin K deficiency did not promote major changes in food intake and energy expenditure, suggesting a peripheral rather than a central effect of cathepsin K. The metabolic phenotype of cathepsin L deficient mice has been investigated as well [27]. Young CatL/ mice have normal body weight until 7 weeks of age, but show markedly reduced body weight gain thereafter, both on low or high fat diet. In high fat fed CatL/mice, body weight reduction occurred in conjunction with reduced fat mass, although food intake and energy expenditure was no less than in wild-type littermates. Reduced levels of serum glucose and insulin and increased glucose tolerance in glucose tolerance testing were also apparent in this model. Interestingly, the improvement of glucose tolerance and serum insulin in CatL/ mice appears be related to an effect of cathepsin L deletion per se, since the phenotype was present in 6-week old CatL/ mice that had not developed significant differences in body weight and fat deposition from control mice. In conclusion, these studies revealed that mice deficient in cathepsin K or in cathepsin L display a lean phenotype and show features of improved glucose homeostasis, partly independent of their reduced fat mass. This beneficial metabolic phenotype persists when mice are challenged by a high fat diet. Thus, in addition to a pro-atherogenic effect, these cathepsins might also contribute to the alterations of glucose and lipid homeostasis seen in obesity. Additionally, since our data in humans point out cathepsin S as clinically relevant, the metabolic phenotype of mice deficient in cathepsin S, that has not been reported so far, could shed new light on the implication of this cathepsin in human obesity. 3.3. Cathepsins K, L and S and adipose differentiation The mechanisms linking cathepsins protease activity to adipose tissue growth are yet to be fully deciphered. Cellular experiments performed in 3T3-L1 murine adipocyte cell line show that cathepsin K gene expression increases over adipose differentiation and that the E64 cathepsin K inhibitor reduces triglyceride accumulation [26]. Other studies investigated the regulation of cathepsins in primary human pre-adipocytes, showing that all three cathepsins K, L and S demonstrate a marked induction after differentiation [27,28,32]. When the human pre-adipocytes were exposed to selective cathepsins inhibitors, they fail to differentiate properly, as evidenced by reduced lipid accumulation. This suggests a pro-adipogenic effect of these proteases, which has been attributed to their capacity to degrade extracellular matrix proteins, mainly fibronectin whose degradation is required for adipogenesis [33]. Altogether, these observations are in line with the lean phenotype of CatK/ and CatL/ mice and suggest that increased cathepsins levels facilitate adipogenesis and/or adipose cell hypertrophy in the adipose tissue of obese subjects, at least in part through extracellular matrix remodelling. 3.4. Mice treated with cathepsin K and cathepsin L pharmacological inhibitors The anti-adipogenic effects of cathepsins deletion in mice and in adipose cell culture experiments prompted to investigate the effect of cathepsins inhibitors in vivo. The phenotypes induced in mice by treatments with cathepsin K [28] or cathepsin L inhibitors [27] are summarized in Table 2. The cathepsin K inhibitor Na-phenoxybenzyloxycarbonyl-L-leucine (2-phenylaminoethyl) amide (K4b) inhibits recombinant human cathepsin K with IC50 < 0.006 mM, whereas it inhibits recombinant human cathepsins S and L with
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IC50 >9.5 and >10 mM, respectively [34]. When administered to high fat fed female mice (ip, 1 mg/kg/day), this inhibitor induced a significant reduction of body weight gain. A similar effect of weight reduction was obtained in genetically obese ob/ob mice that received K4b at 4 weeks of age for 8 weeks. In agreement with the lack of effect of cathepsin K deletion, all tested energy expenditure parameters, including food intake, O2 consumption, CO2 production, and metabolic rate were not affected in wild-type or ob/ob mice by this compound [28]. Of note, K4b might not be very selective in mice. Indeed, a recent study indicates that this compound has a greater potency to inhibit mouse cathepsins S and L than mouse cathepsin K [35]. Therefore, some of the weightreducing effects of 4Kb reported in mice models of obesity might result in part from reduction of cathepsin S and/or cathepsin L activity. The pharmacological inhibition of cathepsin L was performed using CLIK195 on both diet-induced and genetically obese ob/ob mice [27]. Mice were injected intraperitoneally with 100 mg/kg/day of the compound. Compared to vehicle controls, this inhibitor reduced moderately body weight gain in male mice fed a high fat diet and had no significant effect on food intake and glycemic parameters. The same dose of CLIK195, however, demonstrated profound and gender-independent effect in reducing body weight gain, serum insulin levels and glucose intolerance in ob/ob mice, although food consumption and energy expenditure were not substantially altered. If transposable in humans, these data suggest a novel therapeutic strategy for the control of obesity by regulating cathepsin K and/or cathepsin L activities. Because of the known negative effect of cathepsin K on bone formation, several inhibitors have been tested for treating osteoporosis, while cathepsin S inhibitors are in development to treat autoimmune diseases, such as psoriasis, asthma and rheumatoid arthritis [36,37]. Currently, there is no data for their use in countering obesity-linked atherosclerosis and/or metabolic dysfunction in humans. A prerequisite for therapeutic use of pharmacological cathepsins inhibitors is a better understanding of the mechanisms relying cathepsins enzymatic activity to metabolic and inflammatory pathways, specifically in the adipose tissue and at the site of vascular wall alteration. Moreover, it is established that the enzymatic activity of cysteine proteases is regulated by endogenous inhibitors belonging to the cystatin superfamily (see below). How pharmacological inhibition could interfere with endogenous inhibition of cathepsins either locally or systemically, has to be determined. 4. Cystatin C in atherosclerosis The cystatin superfamily comprises a large group of proteins present in a wide variety of organisms, including humans [38e40]. Cystatin inhibitory activity is vital for the regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases, including cysteine cathepsins. Cystatin C, a small protein produced by virtually all organs and found in biological fluids including blood, is the most abundant and potent endogenous inhibitor of cathepsins. Its implication in clinical and experimental atherogenesis has therefore been investigated by several groups. 4.1. Cystatin C in atherogenesis In humans, cystatin C protein is detected in normal arteries, but severely reduced in atherosclerotic and aneurismal aortic lesions where cathepsins K and S are abundant [41]. Moreover, reduced serum cystatin C concentrations were reported in patients with abdominal aortic aneurysms [41,42]. These observations support an
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anti-atherogenic role of cystatin C. Contrary to this hypothesis, epidemiological studies consistently report a positive relationship between elevated circulating cystatin C and cardiovascular outcomes in selected populations, including elderly participants and patients with established vascular diseases or increased cardiovascular risk [43e48]. This counterintuitive association suggests that elevation of cystatin C represents a compensatory mechanism to reduce pro-atherogenic cathepsins activity. 4.2. Cystatin C knockout mice models To assess directly the effect of cystatin C on plaque formation, two groups have investigated the vascular phenotype of double ApoE- cystatin C knockout mice (ApoE //CysC /), with slightly different results (Table 1). Sukhova et al. [49] found that cystatin C deficiency yielded significantly increased tunica media elastic lamina fragmentation and increased SMC accumulation, but no difference in the size of aortic lesions induced by an atherogenic high-cholesterol diet. Of note, collagen content was increased and not reduced, as would be expected after deletion of the cathepsin inhibitor. In the other study, Bengtsson et al. [50] report increased plaque size and macrophages content in the double knockout mice when fed a high fat diet. These findings demonstrate directly the importance of cystatin C in deregulated arterial integrity during experimental atherogenesis. However, the cellular and molecular mechanisms involved, either direct or through cathepsins inhibition, remain to be established. 5. Cystatin C in human obesity Unrelated to its functional role as an inhibitor of cathepsins, cystatin C has emerged as a surrogate marker of the glomerular filtration rate (GFR) at least as good as serum creatinine [51,52]. Indeed, the protein is freely filtered by the renal glomeruli and metabolized by the proximal tubule. However, unlike creatinine, which reflects muscle mass, the main tissue source of cystatin C was unknown. A few studies have reported that increased adiposity or obesity associate with higher cystatin C circulating concentrations. Positive associations of serum cystatin C with BMI were found in a geriatric population [53], in the general population [45], and, with body weight, in a large Caucasian population [54]. Higher waist circumference and increased per cent of body fat also associated with higher serum cystatin C in apparently healthy subjects [55,56]. Recently, a graded association between higher BMI and elevated serum cystatin C was reported in American adults [57]. In these epidemiological studies, however, the contribution of increased adiposity versus reduced GFR in obesity-linked elevation of serum cystatin C was unclear. To address this point, we measured serum cystatin C in a large population of non-obese and obese subjects [58]. Using the abbreviated equation from the Modification of Diet in Renal Disease (MDRD) study [59], we estimated systematically GFR in all participants. This allowed measuring the respective effect of obesity and reduced GFR to increase the circulating concentrations of cystatin C. Interestingly we found that circulating cystatin C was consistently elevated in obese subjects of both genders, independently of reduced eGFR. These observations strongly suggest a role for adipose tissue as a contributor to circulating concentrations of this protein in obesity. In support of this hypothesis, we showed that adipose tissue expression of cystatin C is increased in obesity and the protein abundantly secreted by human adipose tissue explants in vitro, mainly by stroma vascular cells that express high levels of cystatin C. These data suggest that adipose tissue production of cystatin C contributes to the elevation
of circulating concentrations in obesity, although the participation of other cells or tissues where cystatin C is expressed cannot be excluded. The consequences of elevated serum cystatin C in human obesity remain largely hypothetical. Increased serum cystatin C might be part of regulatory mechanisms engaged to control the pro-atherogenic capacity of specific cathepsins. As indicated by the lean phenotype of CatK/ and CatL/ mice, certain cathepsins might promote fat accretion. In this context, increased adipose cystatin C could exert a protective effect to control adipose tissue enlargement through cathepsins inhibition. Although further studies are required to decipher the actual pathophysiological roles of cystatin C, this protein can be added to the list of adiposesecreted factors with the potential to affect adipose tissue biology and obesity-linked complications through local and/or systemic actions. 6. Conclusion In obesity, adipose tissue secretes numerous bioactive molecules that may be acting synergistically in developing cardiovascular diseases and other complications associated with the deregulation of glucose and lipid homeostasis. As reviewed here, members of the cathepsin family, including cathepsins K, L and S, are among the newly discovered potential culprits. This opens new avenues to test whether individual or combined inhibition of selected cathepsins could reduce cardiovascular risk and ameliorate metabolic status in obese subjects. Given the current development of cathepsins inhibitors for diverse pathologies, these drugs could be tested in the near future in obese patients. Nevertheless, further clinical and experimental studies are obviously needed to establish the proof of concept that cathepsins inhibition is beneficial in obesity metabolic and cardiovascular comorbidities. Acknowledgments The authors thanks supports from Clinical Research Contracts (Assistance Publique/Direction de la Recherche Clinique, AOR 02076) for human studies. This work was supported by a grant from the European Community (Collaborative Project ADAPT, HEALTHF262008-201100), a grant from the French National Agency of Research (OBCAT, program ANR-05-PCOD-026-01), a prize DANONE/Fondation pour la Recherche Médicale. References [1] M. Guerre-Millo, Adipose tissue and adipokines: for better or worse. Diabetes Metab. 30 (2004) 13e19. [2] P. Trayhurn, I.S. Wood, Signalling role of adipose tissue: adipokines and inflammation in obesity. Biochem. Soc. Trans. 33 (2005) 1078e1081. [3] P. Trayhurn, Adipocyte biology. Obes. Rev. 8 (Suppl. 1) (2007) 41e44. [4] R. Cancello, C. Henegar, N. Viguerie, S. Taleb, C. Poitou, C. Rouault, M. Coupaye, V. Pelloux, D. Hugol, J.L. Bouillot, A. Bouloumie, G. Barbatelli, S. Cinti, P.A. Svensson, G.S. Barsh, J.D. Zucker, A. Basdevant, D. Langin, K. Clement, Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgeryinduced weight loss. Diabetes 54 (2005) 2277e2286. [5] R. Cancello, J. Tordjman, C. Poitou, G. Guilhem, J.L. Bouillot, D. Hugol, C. Coussieu, A. Basdevant, H.A. Bar, P. Bedossa, M. Guerre-Millo, K. Clement, Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity. Diabetes 55 (2006) 1554e1561. [6] C.A. Curat, V. Wegner, C. Sengenes, A. Miranville, C. Tonus, R. Busse, A. Bouloumie, Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. Diabetologia 49 (2006) 744e747. [7] I. Harman-Boehm, M. Bluher, H. Redel, N. Sion-Vardy, S. Ovadia, E. Avinoach, I. Shai, N. Kloting, M. Stumvoll, N. Bashan, A. Rudich, Macrophage infiltration into omental versus subcutaneous fat across different populations: effect of
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